1. Field of the Invention
The present invention relates to a method for separating antigenantibody complexes from solutions containing free antigens or antibodies. More particularly, the present invention relates to a technique wherein an adsorbent material is used to effect such separation for a multitude of samples without regeneration of the absorbent between samples. This technique is useful in automated immunoassay techniques.
2. Description of the Prior Art
Isotope displacement using specific antibodies to measure minute quantities of insulin was first reported by Yalow et al (Nature 184, 1648) which has led to the widespread practice of this technique for the analysis of a wide variety of biologically important substances. Radioimmunoassay has become a primary technique for the routine analysis of literally hundreds of biochemical and clinically important substances. Radioimmunoassay is now the method of choice for the analysis of many substances, because antibodies with very high selectivity and affinity can be produced which permit measurement of any desired compound in rather impure samples. The amount of impurity can, in many cases, be 10.sup.9 times that of the substance of interest and not interfere with the measurement. This extraordinary selectivity and ability to detect femtomole (10.sup.-15 mole) quantities of substances has pushed the radioimmunoassay to the forefront of modern analytical chemistry.
If the radioimmunoassay has any limitations at all, it is the amount of manual labor and time required to obtain results. A typical assay first involves the combination of the unknown sample or standard, specific isotope tracer and antibody. This solution is then incubated in the cold or at room temperature for at least 20-30 minutes to as long as several days to obtain equilibrium between the antigen (ligand molecule being measured) and the antibody. The antibody bound ligand isotope is then separated from the solution. This is generally accomplished by addition of dextran coated charcoal to absorb the free ligand, by precipitation of the antibody-isotope complex with ammonium sulfate or ethanol or by some other technique such as molecular seive chromatography. The isotope antibody complex is recovered after centrifugation or collection of a specific column fraction and the radioactivity determined usually in an automatic beta or gamma counter. The amount of unknown substance present is determined from standard curves constructed from standards measured at the same time. Increasing additions of unknown sample reduces the specific activity of the isotope tracer thus yielding less radioactivity bound to the antibody.
The manual processing of samples for radioimmunoassay is time consuming, costly and requires meticulous attention to detail. In one laboratory alone, 8000 to 10,000 test tubes per month may be used for radioimmunoassay purposes. The repetitive nature and high precision of these determinations is responsible for considerable variability in the quality and reproducibility of the results. It is obvious that the complete automation of this technique would, or course, be desirable. Several attempts to automate radioimmunoassays have only met with limited success.
The major reason radioimmunoassays (or immunoassays) have not heretofore been readily adapted to automated analysis has been the difficulty in devising on-line methods to separate the antigen-antibody complex from the free antigen or antibody.
For instance, in Johnson, U.S. Pat. No. 3,896,217, a method is provided wherein a sample containing an unknown concentration of a specific antigen, and containing a known concentration of the same antigen tagged with a radioactive isotope, is passed through a bed of an immobilized antibody which is specific in its reactivity for the antigen being detected. As the solution is passed through the bed, both tagged and untagged antigen are bound to the immobilized antibody. It is essential that insufficient antibody is provided in the bed to react with all of the antigen in the solution; the solution passing through the antibody bed will contain both untagged and tagged antigen which is passed into a detector where the amount of unbound, tagged antigen present is measured. The bed must then be washed with a regenerating solvent which extracts all of the bound antigen, and the released antigen is passed into the detector for measurement of the quantity of tagged antigen which has been bound. The results are then correlated to a standard curve for determination of the concentration of untagged antigen in the original sample. This technique however, is not completely satisfactory, since it requires a substantial time delay to effect the necessary extraction of the antigen and regeneration of the immobilized antibody, and consequently, is not completely amenable to rapid analysis, which would be necessary to effect analysis of a multiplicity of samples. In the Johnson technique, it is necessary to use a sufficiently low concentration of immobilized antibody to bind only a portion of the antigen.
Another approach to automated radioimmunoassay has been described by Ertingshausen et al (Clinical Chemistry 21, 1305, 1975). The technique involves the initial automatic pipetting of antibody and antigen reagents followed by precisely timed incubation of the mixed ingredients. Each sample containing the antigen, antibody and the antigen-antibody complex are resolved into a fraction containing the antigen-antibody complex by passing the mixture through individual molecular seive columns, much the way that manual assays are currently performed. One column is thus used for each sample and usually discarded after the analysis. Radioimmunoassays of samples are determined in a static system similar to other conventional techniques, and the concentration of antigen in the original sample is determined by the use of preobtained, standardized curves. This procedure is burdened with much the same manual operational procedures as other prior art techniques which do not permit the continuous and rapid determination of a multiplicity of antigen containing samples.
Alternative approaches involve precipitation or filtration of a double antibody complex (E. Klin. Chem. Klin. Biochem. 13 Jg 1975, 481) or reacting the antigen in an antibody gel (Clin. Chem. 21, 829).
The weakness of all of these methods is that they require a separate separation column or material for each sample being processed. A need therefore has existed for a simple continuous technique to separate the antigen-antibody complex from free antigen or antibody in solution.